Our current corneal research addresses the molecular basis for corneal-specific gene expression, the roles of the abundant, intracellular corneal proteins, the global patterns of corneal gene expression, and stratification of the corneal epithelium. We have reported or accomplished the following this year:[unreadable] [unreadable] Mouse aldehyde dehydrogenase 3a1 (ALDH3A1) comprises half of the water-soluble protein in the corneal epithelial cells of the mouse. We have described the developmental course of corneal expression of this gene. Corneal expression was first detected after birth and high levels were found at postnatal day 14, the time of eye opening and stratification of the epithelial cells. Pax6, Oct1 and p300 were found to synergistically activate the ALDH3A1 promoter, consistent with the refracton hypothesis unifying lens and cornea as a single functional and developmental unit. A position and orientation-dependent negative regulatory element was identified in intron 1 of the gene which represses transcription in corneal and other cells. Multiple DNA constructs have been made containing all intronic and 3 sequences of the ALDH3a1 gene to search for regulatory motifs. These constructs are being examined in transgenic mice. [unreadable] [unreadable] In collaboration with Dr. Jerome Kuszak (Rush Presbyterian Hospital, Chicago), we found reductions in microplicae in the corneal surface epithelial cells of ADLH3a1 null mice, indicative of increased cell sloughing. Microarray data indicated that 50% of the differentially regulated genes in the ALDH3a1 null corneas are those controlled by gamma-interferon. These data suggest that barrier function of the corneal epithelium is compromised causing an immune response. The ALDH3a1 null mice contain Helicobacter (which can lead to ulcers and cancer), while wild type mice do not. Experiments in collaboration with Dr. Vasilis Vasiliou (University of Colorado Health Sciences Center, Denver) using the ALDH3a1 null mice show elevated protein conjugates of 4-hydroxy-nonenal, a substrate of ALDH3A1, consistent with a detoxifying function for this enzyme. One protein, chondromodulin (an anti-angiogenic factor), increases in the cornea of the ALDH3a1 null mice and is located in the cytoplasm and nuclei of the corneal epithelial cells, in contrast to its strictly cytoplasmic presence in wild type mice.[unreadable] [unreadable] We are examining the ocular phenotypes of transgenic mice overexpressing Pax6 and Pax6(5a) transgenes in the cornea. The overexpressing corneas are opaque and enriched with blood vessels. Further studies involving microarray analysis are in progress to identify downstream gene targets of the Pax6 transcription factors. [unreadable] [unreadable] We previously reported that a gelsolin-like protein comprises half of the water-soluble protein of the zebrafish cornea, and that a different gelsolin protein is expressed ubiquitously in the zebrafish at lower levels. In collaboration with Drs. Eugene Koonin (NLM, NIH) and Vasilis Vasiliou (University of Colorado) we established by phylogenetic analysis that the zebrafish corneal-preferred gelsolin-like protein is a scinderin-like protein (also a member of the gelsolin superfamily), while the ubiquitously expressed gelsolin is an authentic gelsolin. Gene duplication has led to 2 scinderin-like genes (scinla and scinlb) and 2 gelsolin genes (gsna and gsnb) in the zebrafish. The scinla protein is corenal preferred, but also expressed in early embryogenesis and contributes to dorsal-ventral signaling. By contrast, the authentic gsna gene is neither corneal preferred nor associated with dorsal-ventral signaling. Thus these closely related genes differ in structure, expression pattern and function. Protein analysis confirmed high expression of actin and scinla, moderate expression of scinlb, and low expression of gsna and gsnb in the zebrafish cornea. A 4 kb promoter fragment including 5 flanking region, exon 1, intron 1 and part of exon 2 of scinla drives the expression of a reporter gene in the embryonic cornea of developing, microinjected zebrafish eggs; expression also occurs in the snout, dorsal fin and tail fin of 3-day-old zebrafish larvae. [unreadable] [unreadable] In the previous fiscal year, KLF4, the most abundant corneal transcription factor identified by SAGE analysis, was deleted specifically in the cornea of mice using floxed genes (KLF4-loxP mice obtained from Dr. Klaus Kaestner, University of Pennsylvania School of Medicine) and Le-Cre mouse (obtained from Dr. Peter Gruss, Max Planck Institute, Germany). The conditionally deleted KLF4 (Klf4CN) mice had a small eye phenotype with defects in lens, cornea and iris. In FY 2005, we identified and characterized multiple phenotypes in the Klf4CN mice ocular surface and identified KLF4 target genes. Even though the embryonic development of the Klf4CN eyes was normal, the postnatal maturation of the Klf4CN cornea was affected as demonstrated by the presence of only 3-4 epithelial cell layers, swollen, vacuolated basal epithelial and endothelial cells, and edematous stroma. The Klf4CN conjunctiva lacked goblet cells and the anterior cortical lens was vacuolated. Expression of the keratin-12 and aquaporin-5 genes was down regulated, consistent with the Klf4CN corneal epithelial fragility and stromal edema, respectively. We have identified a large set of functionally diverse potential KLF4 target genes by microarray analysis, revealing the molecular basis of the wide range of Klf4CN corneal phenotypes . Overall, these results establish KLF4 as an important node in the genetic network of transcription factors regulating corneal homeostasis. These observations provide new insights into the role of KLF4 in post-natal maturation and maintenance of the ocular surface and suggest that the Klf4CN mouse is a useful model for investigating ocular surface pathologies such as dry eye, Meesmann dystrophy and Stevens-Johnson syndrome.[unreadable] [unreadable] SAGE (serial analysis of gene expression) has been performed on the zebrafish cornea in order to characterize gene expression in this tissue. Particular attention is given to identifying corneal transcription factors. In addition, the degenerate eye, skeletal muscle, heart and brain of the blind mole rat has been subjected to SAGE in collaboration with Dr. Eviatar Nevo (University of Haifa, Israel). These blind mole rat experiments are an attempt to identify genes that illuminate new functions for the mammalian eye. SAGE libraries have been made for all these tissues and are presently ready for analysis.[unreadable] [unreadable] Finally, in the previous fiscal year we started exploring the role of microRNAs (miRNAs) in mouse corneal development. miRNAs are a class of small endogenous noncoding RNAs that posttranscriptionally down regulate gene expression and play important roles in diverse processes such as embryonic development, differentiation, and cancer. In association with the RNA-induced silencing complex (RISC), miRNAs either cause target mRNA cleavage and degradation or inhibit mRNA translation. We evaluated corneal miRNA expression profiles by microarray analysis in post-natal day 6 (PN6) and 6-week-old (adult) mice. 42 miRNAs demonstrating at least a 2-fold difference in expression between the two developmental stages were identified. Of these differentially expressed miRNAs, miR-184, miR-31, miR-205, and miR-204 were corneal-enriched and their expression increased in adult corneas compared to PN6 corneas. miR-184 is the most abundant miRNA in adult mouse cornea. The gene for miR-184 is located in an intergenic region on mouse chromosome 9. Preliminary data from 2-dimensional gel electrophoresis corneal proteins indicated differences in protein levels between PN9 and adult corneas. These experiments were discontinued in the last year since the investigator (Dr. Jennifer Pitrak) left the laboratory. However, they are presently being initiated once again.